Our objectives are to isolate and characterize meloanoma associated antigens (MAA). Murine B16 melanoma cells in tissue culture were radiolabelled with H3-leucine, solubilized in non-ionic detergent, and the supernatant precipitated with 50% ammonium sulfate and fractionated on Sephadex G- 200. Three peaks of radioactivity were eluted. Serum of syngeneic mice immunized to melanoma, and serum of rabbit immunized to Bl6 melanoma and absorbed exhaustively with normal murine tissue, bound l0 to 20 times more material in the peak corresponding to a molecular weight of l50,000 than did normal sera. Further absorption of absorbed rabbit antimelanoma sera with normal syngeneic or heterologous murine tissue did not reduce binding of radiolabelled material in this peak, but absorption with equal tissue volume of several unrelated murine tumors including S9l melanoma reduced specific binding by 45-55% and absorption with Bl6 melanoma by 80%. A radioimmunoassay was developed to quantitate the amount of this apparent MAA in tissue. Material crossreacting with MAA was found in lysate of normal murine cells and unrelated tumor cells, but in considerably lesser amount than in Bl6 melanoma. MAA were secreted or shed by melanoma cells in tissue culture. Approximately 20- 40% of intracellular MAA were released in 24 hours, an amount slightly exceeding that of unrelated proteins.